Transforming growth factor receptor III (Betaglycan) regulates the generation of pathogenic Th17 cells in EAE

The transforming growth factor receptor III (TβRIII) is commonly recognized as a co-receptor that promotes the binding of TGFβ family ligands to type I and type II receptors. Within the immune system, TβRIII regulates T cell development in the thymus and is differentially expressed through activation; however, its function in mature T cells is unclear. To begin addressing this question, we developed a conditional knock-out mouse with restricted TβRIII deletion in mature T cells, necessary because genomic deletion of TβRIII results in perinatal mortality. We determined that TβRIII null mice developed more severe autoimmune central nervous neuroinflammatory disease after immunization with myelin oligodendrocyte peptide (MOG35-55) than wild-type littermates. The increase in disease severity in TβRIII null mice was associated with expanded numbers of CNS infiltrating IFNγ+ CD4+ T cells and cells that co-express both IFNγ and IL-17 (IFNγ+/IL-17+), but not IL-17 alone expressing CD4 T cells compared to Tgfbr3fl/fl wild-type controls. This led us to speculate that TβRIII may be involved in regulating conversion of encephalitogenic Th17 to Th1. To directly address this, we generated encephalitogenic Th17 and Th1 cells from wild type and TβRIII null mice for passive transfer of EAE into naïve mice. Remarkably, Th17 encephalitogenic T cells from TβRIII null induced EAE of much greater severity and earlier in onset than those from wild-type mice. The severity of EAE induced by encephalitogenic wild-type and Tgfbr3fl/fl.dLcKCre Th1 cells were similar. Moreover, in vitro restimulation of in vivo primed Tgfbr3fl/fl.dLcKCre T cells, under Th17 but not Th1 polarizing conditions, resulted in a significant increase of IFNγ+ T cells. Altogether, our data indicate that TβRIII is a coreceptor that functions as a key checkpoint in controlling the pathogenicity of autoreactive T cells in neuroinflammation probably through regulating plasticity of Th17 T cells into pathogenic Th1 cells. Importantly, this is the first demonstration that TβRIII has an intrinsic role in T cells.

The transforming growth factor receptor III (TbRIII) is commonly recognized as a co-receptor that promotes the binding of TGFb family ligands to type I and type II receptors. Within the immune system, TbRIII regulates T cell development in the thymus and is differentially expressed through activation; however, its function in mature T cells is unclear. To begin addressing this question, we developed a conditional knock-out mouse with restricted TbRIII deletion in mature T cells, necessary because genomic deletion of TbRIII results in perinatal mortality. We determined that TbRIII null mice developed more severe autoimmune central nervous neuroinflammatory disease after immunization with myelin oligodendrocyte peptide (MOG  ) than wild-type littermates. The increase in disease severity in TbRIII null mice was associated with expanded numbers of CNS infiltrating IFNg + CD4 + T cells and cells that co-express both IFNg and IL-17 (IFNg + / IL-17 + ), but not IL-17 alone expressing CD4 T cells compared to Tgfbr3 fl/fl wildtype controls. This led us to speculate that TbRIII may be involved in regulating conversion of encephalitogenic Th17 to Th1. To directly address this, we generated encephalitogenic Th17 and Th1 cells from wild type and TbRIII null mice for passive transfer of EAE into naïve mice. Remarkably, Th17 encephalitogenic T cells from TbRIII null induced EAE of much greater severity and earlier in onset than those from wild-type mice. The severity of EAE induced by encephalitogenic wild-type and Tgfbr3 fl/fl .dLcKCre Th1 cells were similar. Moreover, in vitro restimulation of in vivo primed Tgfbr3 fl/fl .dLcKCre T cells, under Th17 but not Th1 polarizing conditions, resulted in a significant increase of IFNg + T cells. Altogether, our data

Introduction
Transforming growth factor receptor III (TbRIII), also known as Betaglycan, is a surface proteoglycan that is broadly expressed in many tissues and cell types (1). It is involved in multiple cellular processes including activation, proliferation, adhesion, migration, and organogenesis (2-4); therefore, Tgfbr3 knockout mice die before E18 embryonic stage (5). Most recently, TbRIII has taken relevance in cancer progression, as its loss promotes epithelial-mesenchymal transition and enhances the migratory potential of malignant cells, being proposed as an important marker for metastasis prognosis (6)(7)(8)(9). The TGFb superfamily is composed of more than 30 ligands that can be classified into 3 subfamilies: activins and inhibins, bone morphogenic proteins (BMP's), and TGFbs (including TGFb1, 2, and 3). The canonical pathway triggered by these ligands involves their binding to TbRII and the subsequent recruitment of TbRI, leading to the phosphorylation of R-SMAD proteins and the activation of the common SMAD protein (SMAD4) which is then translocated to the nucleus to regulate gene expression.
TbRIII has been classically described as a co-receptor promoting the binding of all three isoforms of TGFb to TbRII (1,10,11). TbRIII also binds inhibins, BMPs and Fibroblast Growth Factor (2,11). In addition to promoting high affinity binding of TGFb to TbRI and TbRII, TbRIII can also be cleaved from the cell membrane where it binds soluble TGFb, thereby acting as a ligand trap (12). Considering the key role of TGFb and other ligands of the TGFb superfamily in T cell biology (13)(14)(15), it is not surprising that TbRIII has been described as an important regulator of T cell maturation and differentiation. This receptor is important for the survival and differentiation of T cells in the thymus, as fetal thymocytes lacking TbRIII exhibit higher apoptosis and a delay in T cell development (16); therefore it is reasonable to think that TbRIII would function as a regulator of mature T cell proliferation and differentiation. In a previous work, we demonstrated that TbRIII is expressed on T cells, highly expressed on CD4 + T cells (preferentially on naïve and central memory CD4 + T subpopulations) compared to CD8 + T cells, but not on B cells, both in mouse and human (17). Additionally, TbRIII was upregulated after TCR stimulation, with similar expression kinetics to other activation markers such as CD25, CD69, and CD44. Interestingly, both thymic and induced Tregs expressed very low levels of TbRIII, as Foxp3 expression is associated with a decreased expression of TbRIII (17). These data suggest that TbRIII may play an important function during T cell activation and/or during TGFb mediated lineage differentiation.
The activation and differentiation of T cells is a highly regulated process involving several signaling pathways, and under certain circumstances the lack of signal regulation triggers pathologies, such as autoimmune diseases. In this context, TGFb signaling is crucial as TbRI deficiency in CD4 cells in mice results in the development of spontaneous autoimmunity in mice (18). Similarly, when TbRII is deleted in CD4 + T cells, mice develop lethal inflammation and multi-organ autoimmune inflammatory infiltration (19), however the role of TbRIII in autoimmunity is not yet described.
Experimental autoimmune encephalomyelitis (EAE), though imperfect, is the best model to study immunopathogenic mechanisms of human multiple sclerosis (MS) (20,21). CD4 + T cells reactive to myelin antigens initiate the disease and both Th1 and Th17 cells mediate an auto-inflammatory response which results in demyelination and axonal damage in the central nervous system leading to ascending paralysis (22). The developmental of EAE and severity of disease is sensitive to factors that tune thresholds of T cell activation, as well as those that have an impact on differentiation to effector populations. Simply stated, enhanced CD4 + T cell activation, such as attenuation of inhibitory signals or augmentation of costimulation will lead to increased disease severity. Conversely, factors that attenuate T cell activation such as increased inhibitory signals or loss of co-stimulation will decrease EAE severity. Factors that promote differentiation to Th1 cells and pathogenic Th17 cells also enhance EAE severity. IL-6 and TGF-b are key first-step cytokines involved in the differentiation of naïve CD4 + T cells to Th17 cells followed by IL-23 that promotes stability and pathogenicity of Th17 cells (23,24). Additionally, at least in vitro, low concentrations of TGF-b promote generation of inflammatory Th17 while strong TGF-b signaling will shift balance to regulation (13,25). After exposure to certain cytokine (IL-23 and IL-12) cues and signals, Th17 cells can also undergo a process described as plasticity that leads transdifferentiation to IFN-g expressing Th1-like cells through an intermediate that expresses both IL-17 and IFN-g (Th17/1 cells) (26,27). The transdifferentiation promotes pathogenicity in EAE. During this transdifferentiation process, Th17 cells gain the expression of Tbet and may downmodulate expression of the master regulator RORgt (28). Severity of EAE and by extension progression of MS disease will be impacted by molecules that modulate Th17 pathogenicity and transdifferentiation.
To interrogate the function of TbRIII in T cells, we generated a mature T cell specific loss of function mutant mouse. Using this novel mouse, we show that T cell specific TbRIII null mice develop normally, have no obvious alterations in peripheral T cell populations and they respond equivalent to wild type to in vitro activation and CD4 + effector T cell differentiation. Induction of EAE in TbRIII null mice leads to greater severity of disease that is associated with expansion of IFN-g and Th17/1 cells but not Th17 in the central nervous system (CNS). However, encephalitogenic TbRIII null Th17 cells but not Th1 cells are more pathogenic in the context of ability to induce EAE in naïve recipient mice. Overall, our data uncovers a biology of CD4 + T cell specific expression of TbRIII in restraining Th17 pathogenicity.

Mice
Tgfbr3 fl/fl mice were generated at UAB transgenic/ES core facility using ES cells obtained from EUCOMM. B6.Cg-Tg(Lck-icre) 3779Nik/J (dLckCre) were obtained from JAX and bred to Tgfbr3 fl/fl mice to generate Tgfbr3 fl/fl .dLckCre and littermate Tgfbr3 fl/fl mice. Female and male eight-to twelve-week-old littermate mice were used in all the experiments. All animals were bred and maintained in the animal facility at the University of Alabama and the Instituto de Investigaciones Biomedicas (IIB, UNAM, Mexico), in specific pathogen-free conditions, according to the ethical guidelines from the National Institute of Health, the Committee of Animal Care and Use at UAB and the Comitépara el Cuidado y Uso de Animales de Laboratorio (CICUAL) at the IIB UNAM (protocol #176), that describe methods of sacrifice, methods of anesthesia and/or analgesia, and efforts to alleviate suffering.

Assessment of Tgfbr3 deletion
The deletion of Tgfbr3 Exon 5 was assessed by PCR on DNA isolated from T CD4 + cells and compared with CD4-depleted splenocytes using the following primers: forward (TGF5FOR1) TGTTGTGGTGACTGTTGGCA and reverse (TGFEX1FOR1) GTTTCGGAGGGTTCTGTGGT. PCR was performed using the AmpliTaq Gold polymerase (Thermo fisher) following the manufacturer's instructions.
To evaluate the loss of TbRIII expression on peripheral T cells, we used a goat anti-mouse TbRIII antibody followed by incubation with a donkey anti-goat IgG AF488 secondary antibody on live CD4 + , CD8 + , and CD19 + cells. Specifications of antibodies used can be found on Supplementary Material (Table 1) . Secondary antibody (Ab) staining was used as negative control for both thymocytes and splenocytes from Tgfbr3 fl/fl .dLckCre and Tgfbr3 fl/fl mice. Samples were acquired in the NxT cytometer (Thermo fisher) and analyzed with FlowJo X software (BD, San Jose, CA).

Analysis of lymphocyte subpopulations
Spleen, mesenteric lymph nodes, peripheral lymph nodes, and blood samples were obtained from Tgfbr3 fl/fl .dLckCre and Tgfbr3 fl/fl mice. Cells were homogenized, counted, and 1x10 6 cells were stained with different antibody panels to analyze B cells (CD19 + ), T cells (CD4 + and CD8 + ) and among these, naïve T cells (CD44 lo CD62L hi CCR7 + ), central memory (CD44 hi CD62L + CCR7 + ), and effector memory T cells (CD44 hi CD62L -CCR7 -). The complete list of antibodies is shown in Supplementary Material (Table 1). Briefly, antibodies were diluted in PBS and cells stained with 50mL of the antibody mix for 30 min at 37°C (for optimal staining of CCR7). Then, cells were washed with PBS and fixed with a 4% paraformaldehyde solution for 5 min, washed and resuspended in FACs buffer before acquisition. In the case of blood, erythrocyte lysis was performed after staining with ACK buffer. Samples were acquired in the Cytoflex S (Beckman Coulter) cytometer and sample data analysis was done using FlowJo X software (BD).
Activation and proliferation with anti-CD3/ CD28 Dynabeads Naïve T cells (CD4 + CD25 -CD44 lo CD62L hi ) from spleen and peripheral lymph nodes were sorted on the MoFlow FACS sorter and stained with Cell Trace Violet (CTV) following manufacturer's instructions. 5x10 4 CTV + cells per well were cultured in a 96-well plate with Dynabeads ® Mouse T-Activator CD3/CD28 (Invitrogen) at a 2:1 beads/cell ratio and complete RPMI medium for 4 days. Cells were recovered at 12, 24, 48, 72, and 96 hours and stained for the detection of CD4, CD25, CD44, and CD69 and viability. Zombie NIR dye (Biolegend) and antibodies were diluted in PBS and cells were incubated with the mix for 15 min at room temperature, washed, fixed, and acquired in the Attune NxT Cytometer (Thermo Fisher). Data analysis was done using FlowJo X software.

Induction of EAE
Active EAE model was induced as previously described (30). Briefly, mice were immunized with 150 mg of MOG  (GL Biochem Ltd.) emulsified in complete Freund's adjuvant containing 500 mg of Mycobacterium tuberculosis (BD). Mice also received an intraperitoneal injection of 200 ng pertussis toxin (List Biological Laboratories, INC.) immediately following immunization, as well as 2 days after immunization. Mice were scored daily to assess clinical symptoms of EAE for 30 days. The EAE scoring system ranges from a score of 0 to 6, as described below, with higher scores representing increased paralysis and disease severity (31).
The onset of disease presents with a partial loss of tail tone scored as a "1". The disease progresses to a "2" when the tail is completely flaccid and does not respond to stimulation. A score of "3" is accompanied by the paralysis of one hind limb, but not both. A score of "4" is ascribed for complete paralysis of both hind limbs, but the ability to maneuver around the cage and access food and water using the fore limbs. Once a mouse does not have the ability to access food and water, or they have lost over 20% of their baseline body weight, the mouse is scored as a "5" and humanely euthanized. If a mouse is found dead in its enclosure, it is scored as a "6". If a mouse dies during the observation period, or is euthanized, it is removed from analysis the day after death/euthanization.

Passive EAE model
For passive induction of EAE, MOG  reactive Th1 or Th17 cells were prepared as described previously (32,33). Briefly, donor Tgfbr3 fl/fl. dLcKCre and Tgfbr3 fl/fl mice were immunized with 150 µg MOG  in complete Freund's adjuvant containing 500 mg of Mycobacterium tuberculosis (BD). Ten days following immunization, cells from peripheral lymph nodes and spleen were restimulated with 10 mg/mL MOG 35-55 under either Th1 or Th17 polarizing conditions for 3 days. Th1 polarizing conditions consisted of 10 ng/mL IL-12 (Biolegend) and 0.5 mg/mL anti-IL-4 (Biolegend) in IMDM complete culture media. Th17 polarizing conditions consisted of 20 ng/mL IL-6 (Tonbo Biosciences), 20 ng/mL IL-23 (Biolegend), 1 ng/mL TGF-b1 (Tonbo Biosciences), 10 mg/mL anti-IFN-g (Biolegend), and 0.5 mg/mL anti-IL-4 (Biolegend) in IMDM complete culture media. The differentiated cells were harvested, pooled, and dead cells were removed using a Ficoll gradient. These cells were used as "donor cells''. The number of transferred donor cells was normalized based on the percentage of live CD4+ cells; sublethally irradiated (350 rads) naïve mice were injected with 5x10 6 cells (Th1 or Th17) resuspended in 500 mL of PBS via intravenous tail injections. Recipient mice were also intraperitoneally administered 200 ng of the pertussis toxin same as for active EAE and they were scored daily to assess clinical symptoms of EAE for 30 days, as described above (34)(35)(36).

Statistical analysis
A nonparametric Mann-Whitney U test was performed from day 15 to day 30, for the in vivo experiments and independent sample t tests were conducted for in vitro assays. Prism (GraphPad) was used to perform the analysis.

TbRIII deletion in peripheral T cells does not affect the homeostasis of CD4+ and CD8+ cells
To investigate the function of the TbRIII protein in mature T lymphocytes, we bred the Tgfbr3 fl/fl mouse with the distal Lck promoter Cre transgenic mice to generate Tgfbr3 fl/fl .dLckCre mice (37,38). PCR analysis of purified CD4 + T cells from spleen show a prominent 463 bp band, the product after deletion of exon 5, and a faint 583 bp band representing wild type ( Figure 1A). In contrast, CD4 + T cell-depleted splenocytes, predominantly containing B cells and CD8 + T cells, presented the wild type (583 bp) and deleted (463 bp) bands, respectively. We next assessed the expression of TbRIII protein on the cell surface of CD4 + T cells, CD8 + T cells, and CD19 + B cells from peripheral tissue and blood by flow cytometry. On CD4 + T cells from all peripheral tissues (spleen, LN, MLN, and blood), we detected very little expression of TbRIII on CD4 + T cells fromTgfbr3 fl/ fl .dLckCre mice compared to Tgfbr3 fl/fl controls ( Figure 1B and data not shown). The low levels of TbRIII probably represent incomplete deletion that is evident in the PCR results. Although the TbRIII levels on CD8 + T cells from Tgfbr3 fl/fl .dLckCre mice were lower than those from Tgfbr3 fl/fl mice, its expression was clearly detected as compared to secondary Ab control staining. In the thymus, Tgfbr3 fl/fl mice and Tgfbr3 fl/fl .dLckCre had equivalent levels of TbRIII on CD4 + and CD8 + T cells, an expected result since the dLck promoter becomes active only in mature T cells in the periphery (37). A very small population of B cells express Lck and therefore TbRIII expression was mostly unaltered in Tgfbr3 fl/fl .dLckCre mice. Furthermore, B cells express very low levels of TbRIII, as previously reported (17).
TbRIII is differentially expressed on CD4 and CD8 T cell populations with increased expression on naïve and central memory CD4 T cell subpopulations (17), suggesting that TbRIII may be involved in T cell homeostasis. We did not observe any differences in frequencies or total numbers of CD4 + and CD8 + T cells, nor subpopulations of CD4 + T cells in peripheral lymphoid organs or blood ( Figure 2, Supplementary Figure 1). The total number of cells in spleen (SP) and mesenteric lymph nodes (MLN) was similar in Tgfbr3 fl/fl .dLckCre and Tgfbr3 fl/fl mice, however, in peripheral lymph nodes (LN) there was a slight decrease in total cell numbers, as well as CD4 + T cells and CD8 + T cell numbers in Tgfbr3 fl/fl .dLckCre compared to control Tgfbr3 fl/fl mice (Figure 2A). This decrease in LN was primarily within the naïve CD4 and CD8 T cell subsets ( Figure 2B).
TbRIII null CD4 T cell mice develop more severe EAE Autoreactive CD4 + T cells initiate the disease in EAE, the mouse model to study MS (34). Molecules that modulate strength of the CD4 T cell response and/or ability to differentiate into pathogenic effector populations such as Th1, Th17 directly impact both the development and severity of EAE disease (24). Thus, this in vivo model is a robust approach to determine to what extent TbRIII impacts CD4 T cell activation and/or differentiation to effector Th cells. We immunized female and male Tgfbr3 fl/fl .dLckCre and Tgfbr3 fl/fl mice with MOG  and followed the development and progression of EAE over 30 days. We observed that Tgfbr3 fl/fl .dLckCre mice developed EAE with significantly greater severity than Tgfbr3 fl/fl mice, with a median clinical score of 3.28 and 2.76, respectively ( Figure 3A). Tgfbr3 fl/ fl .dLckCre showed greater mortality than Tgfbr3 fl/fl mice from EAE, however, overall survival as assessed by Kaplan-Meier analysis was not significantly different between the two groups (data not shown). It is important to note that the survival analysis only included mice that died spontaneously and none that were euthanized due to severe disease (more than 20% of loss in body weight or clinical score ≥ 3.5). Disease onset was not different between TbRIII null and controls ( Figure 3A).
Th1 and Th17 cells contribute to EAE severity and Treg cells suppress disease (39), therefore we interrogated if increased disease severity in Tgfbr3 fl/fl .dLckCre mice is associated with changes in these effector CD4 T cell populations. Mice were euthanized around the peak of disease, and the T cell populations in the spinal cord were analyzed for frequency of CD4 + T cells expressing IFN-g, IL-17 or  Figure 3B). Although, the total numbers of CD4 + T cells in the spinal cord were not significantly different between Tgfbr3 fl/fl .dLckCre and Tgfbr3 fl/fl mice, TbRIII null mice had significantly higher numbers of Th1 (IFN-g + ) T cells in the spinal cord compared to controls (Supplementary Figure 3). We also performed an analysis of CD25 + Foxp3 + Tregs ( Figure 3B) and CD8 + T cells (not shown) and found no significant differences in the spinal cord of Tgfbr3 fl/ fl .dLckCre compared to Tgfbr3 fl/fl mice with active EAE.
These results indicate that loss of TbRIII leads to expansion of Th effector cells expressing IFN-g in vivo. This inference is supported by an independent experiment showing greater anti-trinitrophenyl (TNP) secondary IgG2c and IgG3, but not IgG1 and IgG2b, antibody response following immunization with TNP-KLH (Supplementary Figure 2). Class switch to IgG2c and IgG3 is promoted by IFN-g expressing CD4 T cells (40).

TbRIII loss does not affect activation and differentiation of CD4+ T cells ex vivo
To investigate the mechanisms underlying the increase in IFNg expressing Th cells contributing to the enhanced EAE and perhaps the IFN-g dependent IgG class switch antibody response observed in TbRIII null mice, we performed a set of functional experiments. We previously reported that expression of TbRIII on CD4 + T cells is induced upon CD3 and CD28 crosslinking, concomitantly with increased expression of CD25, CD69, and CD44 (17), suggesting that this coreceptor could be functionally relevant for T cell activation and differentiation. Therefore, we stimulated CD4 + T cells with anti-CD3/ CD28 and assayed for the induced expression of activation markers including CD25, CD44, and CD69 as a readout for T cell activation. We found no difference in induction and levels of expression of the three activation markers on CD4 + T cells from Tgfbr3 fl/fl .dLckCre and Tgfbr3 fl/fl mice ( Figure 4A). In addition, CD3/CD28 co-stimulation induced T cell proliferation measured by CTV dilution was equivalent between TbRIII null and wild type CD4 + T cells at all time-points between 24 to 96 hours of stimulation ( Figure 4B). This was the case for proliferation index, division index and precursor frequency.
We have previously reported that expression of TbRIII is modulated by T cell activation. Interestingly, TbRIII expression inversely correlates with FoxP3 expression levels during in vitro iTreg differentiation in the presence of TGFb (17). To determine if TbRIII expression is differentially modulated during polarization of CD4 + T cells into Th1 and Th17 cells, we isolated naïve CD4 + T cells and cultured them under appropriate skewing conditions. We observed that TbRIII expression was higher on CD4 + T cells cultured in either Th1 or Th17 polarizing conditions than nonskewed CD4 + T cells ( Figure 5A). Furthermore, expression of TbRIII was greater on Th17 skewed cells compared to Th1 skewed cells. Focusing on only cytokine-expressing cells, we observed that IL-17 + and not IFN-g + expression was associated with enhanced upregulation of TbRIII expression. This led us to hypothesize that TbRIII might contribute to Th17 differentiation more than Th1 differentiation. However, in ex vivo Th differentiation cultures, naïve CD4 + T cells from Tgfbr3 fl/fl and Tgfbr3 fl/fl .dLckCre mice differentiated similarly under Th1 and Th17 skewing conditions ( Figure 5B). There was a trend towards increase in IFN-g + cells in TbRIII null T cells, but the difference was not significant. We tested if TbRIII impact on Th differentiation is dependent cell division using CTV dilution assay. Here again we were unable to identify any difference between Tgfbr3 fl/fl .dLckCre and Tgfbr3 fl/fl CD4 + T cells ( Figure 5B). Overall, these results indicate that under conventional ex vivo culture conditions, the activation and differentiation of naïve CD4 + T cells is not altered by the presence or absence of TbRIII.
TbRIII null encephalitogenic Th17 cells induce more severe EAE As previously described, independent and overlapping cytokinemediated mechanisms regulate pathogenicity of encephalitogenic Th1 and Th17 cells in their ability to passively induce EAE in naïve mice (32,41,42). Relevant to this, increased severity of EAE in TbRIII null mice was associated with a greater proportion and numbers of IFN-g expressing Th cells in spinal cords ( Figure 3). This led us to predict that TbRIII likely restrains pathogenicity of effector Th cells, particularly Th1 cells. To test for this, we generated encephalitogenic Th17 or Th1 cells by immunizingTgfbr3 fl/ fl .dLckCre mice or Tgfbr3 fl/fl mice, isolated in vivo primed/activated T cells and subsequent ex vivo antigen (MOG  ) induced expansion in the presence of Th17 or Th1 skewing conditions. These encephalitogenic Th1 or Th17 cells were transferred into naïve mice and progression of EAE was determined. Recipients of TbRIII null Th17 cells developed EAE with earlier onset, beginning day 10, vs controls beginning day 12 ( Figure 6A). TbRIII null Th17 cells also induced EAE with much greater severity than controls. There was no mortality in either of the recipient groups. In contrast to Th17 transfers, there was no difference between wild type and TbRIII encephalitogenic Th1 cells to induce EAE and the majority of recipients died within the 30-day time period (Figure 6B). We characterized the encephalitogenic Th17 and Th1 cells that were in vivo primed, and ex vivo restimulated/skewed. We determined that A B FIGURE 3 T cell specific TbRIII null mice develop more severe EAE after immunization with MOG  . Mice with an EAE clinical score above 4 or a drop in weight of over 20%, mice were humanely euthanized. After a mouse was euthanized, it ceased to be scored and was therefore removed from future analysis. In (B) each dot represents an individual mouse. Data is mean ± SEM; Nonparametric t-test for (A) and parametric t-test for (B). **=p<0.01,, ****=p<0.0001.
CD4 + T cells from TbRIII null mice skewed under Th17 conditions with MOG  had an expanded proportion of IFN-g expressing cells but equivalent IL-17 + cells to controls ( Figure 6C upper graph). There was no difference between TbRIII null and controls for cells skewed under Th1 conditions ( Figure 6C bottom graph). Overall, these data indicate that TbRIII restrains the pathogenicity of encephalitogenic Th17 cells likely by a mechanism regulating the transition to IFN-g expressing Th cells.

Discussion
TbRIII is expressed on CD4 + and CD8 + T cells at all stages of development, however its role in these populations has been elusive. Our group previously demonstrated from experiments in fetal thymic organ cultures that TbRIII plays a protective role in thymocyte survival. Furthermore, we showed that the expression of the coreceptor is upregulated upon T cell activation and therefore we hypothesized that TbRIII modulates processes downstream of TCR signaling. However, the ability to examine the function of TbRIII has been a challenge, as this coreceptor plays a key role in several developmental processes during fetal life such that its developmental knockout results in neonatal mortality (5). To overcome this limitation, we generated a Tgfbr3 fl/fl mouse and bred it with the dLck.Cre mouse for conditional deletion of TbRIII in mature T cells (37). We confirmed that Cre-mediated deletion was very effective by PCR analysis of genomic DNA for Cre-mediated deletion of Tgfbr3 exon 5. In CD4 + T depleted spleen cells, two bands were observed: the lower band likely corresponds to CD8 + T cells (delete exon 5), and the upper band attributable to B cells (wild type band). CD4 + T cells were not further segregated into subpopulations since they all express high levels of TbRIII (17).
Flow cytometry analysis confirmed that TbRIII expression was selectively abrogated in CD4 + T cells ( Figure 1B) and moderately on CD8 + T cells, but not in thymic CD4 + T cells and CD19 + B cells, as the Lck distal promoter is selectively active in late mature T cells. Tgfbr3 exon 5 deletion did not result in complete protein ablation, and although we cannot exclude that the remaining expression of TbRIII is functional, its dramatic decrease in expression observed in Tgfbr3 f/ f .dLckCre mice was sufficient to increase the severity of EAE induced by active immunization with MOG 35-55 peptide or by passive immunization with encephalitogenic Th17 cells.
Although we did not find significant differences in the proportion of T cell subsets between Tgfbr3 fl/fl .dLckCre and Tgfbr3 fl/fl mice, there was a trend towards a decrease in LN CD4 + and CD8 + T cell numbers, which was not present in spleen and MLN, suggesting a possible LNspecific homing defect. Consistent with our previous report, TbRIII was differentially expressed between subpopulations of T cells, with greater levels on CD4 + compared to CD8 + T cells (17). This is especially relevant given that this receptor modulates cell migration and metastasis in different cancer type cells (3). We have previously shown that TbRIII is upregulated upon TCR crosslinking in concomitantly to other activation markers, such as CD69, CD25 and CD44 (17). Therefore, we speculated that this coreceptor might be involved in T cell activation. However, we found that under nonskewing conditions, T cell activation was not significantly affected by the absence of TbRIII.
Given the role of TbRIII in the modulation of TGFb signaling (43) and our recent report showing that the expression of TbRIII is decreases with increase in Foxp3 expression during induced-Treg (iTreg) culture conditions (17), we predicted that this correceptor may modulate differentiation of naive CD4 T cells to Th17 and/or Th1 effector cells. The differentiation of naive CD4 + T cells following under appropriate polarizing conditions induced greatly elevated expression of TbRIII on Th17 cells, at levels greater than that on Th1 cells. However, the loss of TbRIII had no effect on the ability of naive T cells to differentiate into Th1 and Th17 under in vitro polarizing conditions. It is important to appreciate that TbRIII interacts with several different molecules; these include activins, inhibins, bone morphogenic proteins, in addition to TGFbs (1). The in vitro culture conditions do not replicate any of these interactions and therefore does exclude the possibility that TbRIII plays a role in Th1 and Th17 functional activities in vivo. In autoimmune diseases, including MS and its mouse model EAE, the importance of Th1 and Th17 cells in the pathogenesis of disease is well established (32). We found that TbRIII null mice developed more severe EAE than littermate controls. The severity of EAE was associated with an expanded proportion of Th1 and Th17/1 (IFN-g + IL-17 + ) cells but not Th17 cells. The Th17/1 cells represent Th17 cells undergoing transition from Th17 to Th1, a process defined as plasticity (44). We and others have shown that these Th17/1 cells likely contribute more to pathogenicity in MS and EAE (45)(46)(47). The expanded Th1 cells in the spinal cord of TbRIII null mice may represent a function of TbRIII to restrict the differentiation of naïve CD4 + T cells into Th1 cells. This concept is supported by the observation that class switch of antibody to IFNg-dependent isotypes (IgG2c and IgG3) was significantly greater in TbRIII null mice, although it was moderately observed for IgG1 and IgG2b.
Passive transfer of in vivo generated, ex vivo expanded encephalitogenic Th17 cells from TbRIII null mice induced significantly greater EAE than controls. The enhanced disease was not due to differences in numbers of transferred Th17 cells, but there was a small but significantly greater number of IFN-g expressing cells generated from TbRIII null donors under Th17 conditions. It is unlikely that the increased pathogenicity is merely due to the presence of IFN-g expressing Th cells as we did not observe any difference for Th1 transfers between TbRIII null and controls. This indicates to us that in the absence of TbRIII, T cells receiving Th17 environmental cues are more pathogenic. It is well appreciated that specific cues can shift the pathogenicity balance of Th17 cells (44,48,49).
The original view of the function of TbRIII as merely an accessory coreceptor for augmenting signals from TbRII-TbRII heterodimer A B FIGURE 5 TbRIII on Th1 and Th17 cells in vitro differentiated CD4 + T cells. The expression of TbRIII was evaluated on CD4 + T cells under Th1, Th17 and non skewing (N.S) conditions. (A) TbRIII expression on CD4 + T cells under non skewing, Th1 and Th17 conditions. Representative histograms TbRIII expression on Th1, Th17, NS and secondary Ab control (left). Graphs of the increased TbRIII expression relative to non skewing conditions on CD4 + , CD4 + IFNg + and CD4 + IL17 + T cells (right). (B) Representative dot plots of IFN-g + CTV + and IL-17 + CTV + live CD4 + T cells following in vitro Th1 and Th17 differentiation from Tgfbr3 fl/fl .dLcKCre (red) and Tgfbr3 fl/fl (blue) mice; below are the graphs of IFN-g or IL-17 increased percentage and median fluorescence relative to non skewing compared to Th1 and Th17 skewing conditions. Bar graphs show mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. n =5 independent experiments for all conditions ****p<0.0001. needs to be reevaluated. TbRI or TbRII deficiency in CD4 cells results in the development of spontaneous autoimmunity in mice (18)(19)(20). If the main role of TbRIII is to function as a co-receptor for TbRI and TbRII, we would expect mice lacking TbRIII on CD4 + T cells to also exhibit spontaneous autoimmunity and death. However, we have studied theTgfbr3 fl/fl .CD4Cre mouse, which lacks TbRIII in all CD4 + T cells, and this mouse does not develop spontaneous autoimmunity up to 18 months of age, data not shown. Additionally, when we crossed this mouse with a dLckCre mouse to generate a Tgfbr3 fl/ fl .dLckCre mouse, which deletes TbRIII on mature T cells, no hyperactivation was observed as reported for TbRII null mice generated in a similar (38).
Results showed that TbRIII in T cells functions to restrict the development of pathogenic effector cells necessary for disease progression and severity in MOG  immunization induced active EAE disease. This is evident by the fact that Tgfbr3 fl/fl .dLckCre mice developed significantly more severe EAE than Tgfbr3 fl/fl mice by both immunization with MOG  and induction via the passive transfer of Th17 polarized T cells. The increase in disease severity in Tgfbr3 fl/ fl .dLckCre mice was also associated with an increased number and proportion of IFN-g producing CD4 + T cells present in the spinal cord. However, there was no difference in IL-17 positive cells in the spinal cord of mice with EAE.
The passive transfer of Th1 polarized cells, regardless of the presence of TbRIII on T cells, resulted in rapid onset of severe disease and high mortality. This high level of mortality and disease was present despite the relatively low level of pathogenic T cell populations seen in culture prior to transfer compared to Th17 transfers. Additionally, the same level of sub-lethal radiation was applied to both Th1 and Th17 recipients and there was no mortality observed in Th17 transfer recipients. This may suggest a high level of pathogenic expansion in the Th1 passive transfer recipient mice regardless of the presence of TbRIII (50). The in vitro MOG restimulation/polarization data also suggests that TbRIII has no effect on the ability of naïve T cells to polarize towards Th1. Although the possibility cannot be excluded entirely, EAE induced by passive transfer of encephalitogenic Th1 cells f r o m T b R I I I s u ffi c i e n t a n d T b R I I I n u l l m i c e w e r e indistinguishable even when the numbers of T cells transferred were reduced to as low as 2x10 6 cells (data not shown). This indicates to us that the role of TbRIII in Th1 generation and/or pathogenicity may be subtle or needs to be addressed with other disease models.
The passive transfer Th17 EAE experiments showed that naïve recipient mice developed more severe disease after being injected with MOG-specific CD4 + T cells lacking TbRIII which had undergone Th17 polarization. However, investigation of these cells prior to transfer suggests that the primary cytokine produced by Th17 cells, IL-17, is likely not responsible for the difference in disease. The percentage of IL-17 producing cells was no different in cells lacking TbRIII and wild type in Th17 polarized MOG-restimulated cultures. Instead, MOG-specific CD4 + T cells lacking TbRIII which had undergone Th17 polarization produced a significantly higher proportion of the cytokine IFN-g than cells from donor wild type mice. This supports our data showing increased IFN-g expressing cells in the spinal cord of mice with active EAE. These results suggest that MOG restimulated Th17 polarized CD4 + T cells lacking TbRIII may have undergone what is known as plasticity. Th17 cells with appropriate cytokine signals, such as IL-23 and IL-12 can transdifferentiate into IFN-g producing Th1 cells (51). During conversion, the IL-17 expressing CD4 + T cells go through an intermediate stage expressing both IFN-g and IL-17; in fact, it was observed that TbRIII null EAE mice had a greater proportion of the double cytokine producing cells than TbRIII sufficient mice [31]. Th1 cells generated from Th17 cells are also defined as ex-Th17 cells (27,52,53). Other effector populations such as Th1 and Th2 cells are fixed in their lineage and are thought to be unable to undergo this process of change to other effector cell populations (27). When Th17 cells begin to downregulate the expression of the master regulator RORgt and begin to upregulate the master regulator T-bet, they begin to take on the profile of Th1 cells and produce IFN-g. The transdifferentiation of Th17 to Th1 cells is important for normal immune system function, such as promoting anti-tumor growth (54). However, there is evidence that suggests Th17 cells that transdifferentiate into Th1 cells can be particularly pathogenic. For example, the so-called ex-Th17 cells that produce IFN-g are thought to be the main drivers of intestinal pathology in humans with inflammatory bowel disease and colitis (55,56). Additionally, evidence suggests that ex-Th17 cells produce higher levels of cytokines than classical Th1 or Th17 cells and are also resistant to Treg suppression of proliferation and cytokine production (53).

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The animal study was reviewed and approved by the "Comité Interno para el Cuidado y Uso de Animales de Laboratorio, (CICUAL), Protocol #176 at the Biomedical Research Institute, UNAM, and the UAB Institutional Animal Care and Use Committee (IACUC).

Author contributions
GS and CR contributed to conception and design of the study. SOF, ROA, and SJD wrote the first draft of the manuscript. SOF, SJD, CMS, NAD, SMO, MSL, RC, PDS, NY, and ASL performed the experiments. SOF, ROA, and SJD organized the database and analyzed data. GS and CR wrote the final version of the manuscript. All authors contributed to the article and approved the submitted version.

Funding
This work was supported by a Grant from Universidad Nacional Autońoma de Mexico DGAPA PAPIIT IN213319. CR was supported from NIH Grants R21 MD015319, R21 AI107748. UAB Flow Cytometry and Single Cell Core Facility was supported by AI027767 and CA013148.